Granulocyte-colony stimulating factor (G-CSF) is a cytokine that stimulates stem cells of bone marrow and leukocytes to induce them to differentiate and proliferate. It is a glycoprotein ranging in molecular weight from 18,000 to 19,000 Da with a pI of 6.1 (5.5-6.1 depending on the degree of glycosylation (Nomura et al., EMBO J. 5(5): 871-876, 1986).
Recombinant DNA technology discovered the molecular and genetic properties of G-CSF (Clark and Kamen, Science, 236:1229-1237, 1987). Since the cloning of human G-CSF gene from the cDNA libraries constructed with mRNAs isolated from CHU-2 and human bladder carcinoma 5637 cell lines (Nagata et al., Nature, 319: 415-418, 1986; Nagata et al., EMBO J., 5(3): 575-581, 1986; Souza et al., Science, 232: 61-65, 1986), recombinant DNA technology has allowed G-CSF to be produced from mammal cells and prokaryotes. In addition, the present inventors have found that a modified hG-CSF, which is different from the wild-type in that at least one amino acid residue, especially the cysteine residue at position 17 is substituted with a different amino acid residue, can be secreted in a form free of methionine residues at the N-terminus thereof on a large scale into a periplasm (Korean Patent No. 10-356140).
Since polypeptides tend to easily denature due to their low stability, be degraded by proteolytic enzymes in the blood and easily passed through the kidney or liver, protein medicaments, including polypeptides as pharmaceutically effective components, need to be frequently administered to patients to maintain the desired blood level concentrations and titers. However, this frequent administration of protein medicaments, especially by injection, causes pain in patients.
To solve these problems, a lot of effort has been put into improving the serum stability of protein drugs and maintaining the drugs in the blood at high levels for a prolonged period of time, and thus maximizing the pharmaceutical efficacy of the drugs. For use in long-acting formulations, protein drugs must be formulated to have high stability and have their titers maintained at sufficiently high levels without incurring immune responses in patients.
To stabilize proteins and prevent enzymatic degradation and clearance by the kidneys, a polymer having high solubility, such as polyethylene glycol (PEG), was conventionally used to chemically modify the surface of a protein drug. By binding to specific or various regions of a target protein, PEG stabilizes the protein and prevents hydrolysis, without causing serious side effects (Sada et al., J. Fermentation Bioengineering 71:137-139, 1991). However, despite its capability to enhance protein stability, PEGylation has problems such as greatly reducing the titers of physiologically active proteins. Further, the yield decreases with increasing molecular weight of the PEG due to the reduced reactivity of the proteins.
An alternative method for improving the in vivo stability of physiologically active proteins is by linking a gene of a physiologically active protein to a gene encoding a protein having high serum stability by genetic recombination technology and culturing the cells transfected with the recombinant gene to produce a fusion protein. For example, a fusion protein can be prepared by conjugating albumin, a protein known to be the most effective in enhancing protein stability, or its fragment to a physiologically active protein of interest by genetic recombination (PCT Publication Nos. WO 93/15199 and WO 93/15200, European Pat. Publication No. 413,622).
Another method is to use an immunoglobulin. As described in U.S. Pat. No. 5,045,312, human growth hormone is conjugated to bovine serum albumin or mouse immunoglobulin by use of a cross-linking agent. The conjugates have enhanced activity when compared with unmodified growth hormone. Carbodiimide or glutaraldehyde is employed as the cross-linking agent. Non-specifically bonding to the peptides, however, such low-molecular weight cross-linking agents do not allow the formation of homogeneous conjugates and are even toxic in vivo. In addition, the patent shows activity enhancement only thanks to chemical coupling with the growth hormone. The method of the patent cannot guarantee activity enhancement to various kinds of polypeptide drugs, so that the patent does not recognize even protein stability-related factors, such as duration, blood half-period, etc.
Recently a drug formulation has been suggested that is a long-acting protein drug formulation with improvement in both in vivo duration and stability. For use in the long-acting drug formulation, a protein conjugate is prepared by covalently linking a physiologically active polypeptide, a non-polypeptide polymer and an immonoglobulin Fc fragment (Korean Patent No. 10-0567902 and 10-0725315).
In this method, G-CSF can be used as a physiologically active polypeptide to afford a long-acting G-CSF conjugate. To apply long-acting G-CSF conjugates to drug products, it is necessary to maintain the pharmaceutical efficacy thereof in vivo while restraining physicochemical changes such as light-, heat- or additives-induced degeneration, aggregation, adsorption or hydrolysis during storage and transportation. Long-acting G-CSF conjugates are more difficult to stabilize than an G-CSF polypeptide itself because they are increased in volume and molecular weight.
Generally, proteins have a very short half life and, when exposed to unsuitable temperatures, water-air interfaces, high pressures, physical/mechanical stress, organic solvents, microbial contamination, etc., they undergo such degeneration as the aggregation of monomers, precipitation by aggregation, and adsorption onto the surface of containers. When degenerated, proteins lose their physicochemical properties and physiological activity. Once degenerated, proteins almost cannot recover their original properties because the degeneration is irreversible. Particularly in the case of the proteins that are administered in trace amounts of hundreds migrograms per injection, such as G-CSF, when they lose stability and thus are absorbed onto the surface of the container, a relatively great amount of damage results. In addition, absorbed proteins easily aggregate during a degeneration process, and aggregates of the degenerated proteins, when administered into the body, act as antigens, unlike proteins synthesized in vivo. Thus, proteins must be administered in a stable form. Many studies have been done to prevent the degeneration of proteins in solutions (John Geigert, J. Parenteral Sci. Tech., 43(5): 220-224, 1989; David Wong, Pharm. Tech., 34-48, 1997; Wei Wang., Int. J. Pharm., 185: 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25: 267-340, 1986; Michelle et. Al., Int. J. Pharm. 120: 179-188, 1995).
Lyophilization is applied to some protein drugs to achieve the goal of stability. However, lyophilized products are inconvenient in that they must be re-dissolved in injection water for use. In addition, they need massive investment on large-capacity freeze-driers because lyophilization process is included in the production processes thereof. The confrication of proteins by use of a spray drier was suggested. However, this method is economically unfavorable due to low production yield. Further, a spray-drying process exposes the proteins to high temperatures, thus having negative influences on the stability of the proteins.
As an alternative to overcome the limitations, stabilizers have appeared that, when added to proteins in solution, can restrain physicochemical changes of protein drugs and maintain in vivo pharmaceutical efficiency even after having been stored for a long period of time. Among them are carbohydrates, amino acids, proteins, surfactants, polymers and salts. Inter alia, human serum albumin has been widely used as a stabilizer for various protein drugs, with certification for its performance (Edward Tarelli et al., Biologicals, 26: 331-346, 1998).
A typical purification process for human serum albumin includes inactivating biological contaminants such as mycoplasma, prion, bacteria and virus or screening or examining one or more biological contaminants or pathogens. However, there is always the risk that patients are exposed to the biological contaminants because they are not completely removed or inactivated. For example, human blood from donators is screened to examine whether it contains certain viruses. However, this process is not always reliable. Particularly, certain viruses existing in a very small number cannot be detected.
Alternatives to human serum albumin have recently been suggested, including recombinant albumin (Korean Patent Laid-Open Publication No. 10-2004-0111351) and albumin-free G-CSF (Korean Patent Nos. 10-0560697 and 10-0596610).
Although employing stabilizers free of albumin, different proteins may be gradually inactivated due to the chemical differences thereof because they are subjected to different ratios and conditions during storage. The effect of a stabilizer on the storage term of proteins differs from one protein to another. That is, various stabilizers may be used at different ratios depending on physicochemical properties of the proteins of interest.
In addition, different stabilizers, when concurrently used, may bring about reverse effects due to competition and the erroneous operation thereof. A combination of different stabilizers also elicits different effects because they cause the proteins to change in characteristics or concentration during storage. Because each stabilizer suitably performs its stabilizing activity in a certain range of concentrations, many efforts must be made to combine the kinds and concentrations of different stabilizers, with care.
Particularly, as for long-acting G-CSF conjugates which are improved in in vivo duration and stability, their molecular weights and volumes are quite different from those of general G-CSF compounds because they are composed of the physiologically active peptide G-CSF and immunoglobulin fragment Fc. In addition, the stability of immunoglobulin Fc fragments varies highly depending on pH. Thus, conventional stabilizers for G-CSF cannot be employed as they are. Accordingly, stabilizers with special compositions different from those of stabilizers for G-CSF are required for long-acting G-CSF conjugates.
Leading to the present invention, intensive and thorough research into the development of the development of a stable liquid formulation for long-acting G-CSF conjugates, capable of retaining pharmaceutical efficacy for a long term without viral infection, resulted in the finding that a stabilizer comprising buffer in a certain pH range and a highly concentration of mannitol endows long-acting G-CSF conjugates with enhanced stability and allows the formation of economical and stable liquid formulations of long-acting G-CSF conjugates.